The present invention relates to a method of production of transgenic plants from meristems, the said plants being wholly transformed in the T0 generation. The invention also relates to the transformed explants obtained during the method.
Genetic engineering techniques are now commonly applied in the breeding of plant species. These techniques allow the introduction of new characters which are difficult or impossible to introduce by conventional techniques. In spite of the development of these techniques, there are however certain species which cannot be easily subjected to transformation or which cannot be regenerated from differentiated explants such as cotyledons or leaves. For these species (for example sunflower, cotton, pea, bean, soybean and the like), it has been demonstrated that these difficulties could, in part, be overcome using, as explant for the transformation, a meristematic explant, for example an apical meristem.
The use of meristems allows the regeneration of fertile plants without a callus formation stage. The technique of regeneration from meristems applies to a large number of different species and, within the same species, to a large number of genotypes. By virtue of this technique, it has been possible for numerous recalcitrant species to be transformed and regenerated (see for example: Bidney et al., Plant Molecular Biol. 18, 301-313, 1992; Bidney et al., Proc. 13e Int. Sunflower Conf. Vol II, Pisa Sept. 1992; Schrammeijer et al., Plant Cell Rep. 9: 55-60, 1990; Christou et al., The Plant Journal, 2(3), 283-290, 1992; Gambley et al., Plant Cell Rep. 12: 343-346, 1993; Russel et al., Plant Cell Rep. 12: 165-169, 1993).
This method has, however, some disadvantages. The transformation frequency is extremely low and the regenerated plants are almost exclusively chimeric, that is to say that some tissues are transformed while others are not. This characteristic results from the fact that the meristem produces the cellular material at the origin of all the tissues of the various plant organs (stem, roots, leaves). Now, regardless of the technique used, the transformation operation only leads to the transformation of a limited number of cells within the explant, these cells being distributed randomly. The meristem is never wholly transformed following such a manipulation. The plants regenerated from a meristem having undergone a transformation stage are therefore chimeric. Wholly transgenic plants are obtained only in the progeny and provided that the germinal line cells have been affected by the transformation. This type of process is described for example in: Bidney et al., Plant Molecular Biol. 18, 301-313, 1992; Schrammeijer et al., Plant Cell Rep. 9: 55-60, 1990.
The disadvantages presented by the production of chimeric plants are recognized in the art, but it has not been possible to offer any solution. A system of labelling which makes it possible to recognize, among the T0 chimeric plants, those which had undergone transformation of the germinal line and which were therefore capable of transmitting the new character to their progeny, has been described (Christou et al., The Plant Journal, 2(3), 283-290, 1992). This method facilitates the production, in the next generation, of wholly transformed plants, but the plants produced in the T0 generation are still chimeric.
Up until now, no method exists which makes it possible to produce in a systematic and predictable manner, transgenic plants, wholly transformed in the T0 generation, from meristematic explants.
The present invention solves this technical problem. The invention is based on the development, by the inventors, of a method which makes it possible to enrich the meristems in transformed cells, in order to arrive at wholly transformed meristems, that is to say meristems all of whose cells are transformed. According to the invention, the regeneration of plants is then carried out exclusively from these wholly transformed explants, which will guarantee the transgenic character of the plants obtained. The inventors have also demonstrated that, under certain conditions, the new formation of newly formed leaf meristems and buds on the leaves of explants derived from meristems can be deduced. The existence of these newly formed leaf buds, containing newly formed leaf meristems, has never been described in the literature. The method of the invention also allows the production of these newly formed leaf meristems in a wholly transformed state. They constitute, in this case, an excellent cellular material for the regeneration of wholly transformed plants in T0.
In general terms, the invention therefore provides a method of production of wholly transformed meristems, and a method which makes it possible to regenerate exclusively from these explants wholly transgenic plants in T0.
More particularly, the invention relates to a method of production of transgenic plants, wholly transformed in the T0 generation, comprising:
a) a stage for the genetic transformation of a meristematic explant, and
b) a stage for selective culture which allows the specific development, among all the transformed cells, of those which are at the origin of the secondary meristems and/or of those capable of giving rise to newly formed leaf meristems;
c) the regeneration, from the cellular material obtained during stage ii), of transgenic plants.
Within the context of the present invention, the term xe2x80x9cmeristematic explantxe2x80x9d means an explant consisting essentially or exclusively of meristematic tissue or of tissue capable of becoming, during its development, meristematic. According to the invention, this term covers especially:
an apical meristem (also known in the art as xe2x80x9cprimary meristemxe2x80x9d), particularly an apical stem meristem, that is to say at the origin of the stem;
a newly formed leaf bud;
part of a young leaf capable of giving rise to newly formed leaf buds.
The term xe2x80x9cnewly formed leaf budxe2x80x9d means a bud carried on the upper epidermis of a leaf. It contains a newly formed leaf meristem. Its development is induced xe2x80x9cartificiallyxe2x80x9d by a series of precise culture conditions.
The nature of these various explants will be described later.
The term xe2x80x9csecondary meristemxe2x80x9d means a meristem derived from a primary meristem. In particular, this term relates, within the context of the invention, to the axillary meristems situated at the axil of the leaves and responsible for the construction of the lateral branches of the plant.
The term xe2x80x9caxillary budxe2x80x9d means a bud situated at the axial of a leaf, containing a secondary meristem.
The term xe2x80x9cnewly formed leaf meristemxe2x80x9d means a meristem contained in a new formed leaf bud. The formation of these meristems is caused xe2x80x9cartificallyxe2x80x9d by culture conditions which will be defined later.
At the cellular level, the secondary meristems and the newly formed leaf meristems have a structural organization and an action which are identical to those of the principal meristem, but are smaller in size. Moreover, the newly formed leaf meristems are smaller than the secondary meristems.
The studies leading to the development of the present invention are based on the following morphogenetic principles. The apical meristem contains, at the axil of the leaf Primordia, cells which, in a predetermined manner, will give rise, by cell multiplication, to secondary meristems. These xe2x80x9cpreprogammedxe2x80x9d cells could be transformed during a genetic transformation procedure. The secondary meristems derived from the multiplication of these cells will be composed exclusively of transformed cells. The same is true of the cells which, under favourable culture conditions, will give rise to newly formed leaf meristems. Consequently, the plants regenerated from these transformed secondary meristems and from transformed newly formed leaf meristems will be wholly transgenic.
The inventors have developed a selective culture system which favours the development, among the transformed cells, of those which are at the origin of the secondary meristems and of the newly formed leaf meristems. The other cells will be eliminated.
Preferably, the selective culture comprises the following stages:
i) the culture, on a selective medium, of the meristematic explant having undergone the transformation stage;
ii) the removal of the transformed axially buds and optionally of the transformed newly formed leaf buds, which are obtained during stage i);
iii) the culture, on a selective medium, of the transformed buds obtained according to ii);
iv) repetition, at least one, of stage ii) and iii).
In other words, according to the invention, the explant having undergone the transformation stage is cultured on a selective medium, for a determined time. When shoots and a few leaves appear, the culture is interrupted and the axillary buds and, where appropriate, the newly formed leaf buds, which are at least partially transformed, are removed. The transformed state is recognized by means of the selective marker. The buds are then transplanted and cultured on a selective medium until they produce, in turn, axillary buds and optionally newly formed leaf buds. The culture is interrupted and the transformed buds are again removed and cultured. By repeating this xe2x80x9cculture of limited duration/removal of the budsxe2x80x9d cycle several times, wholly transformed buds are obtained, in fact, with each cycle, the number of transformed cells in the bud and the proportion of transformed cells relative to the non-transformed cells increases by virtue of cell multiplication. The cycles are therefore repeated until wholly transformed buds are obtained. A wholly transformed bud is recognized by its capacity to produce a shoot which has, throughout, a green colour characteristic of its capacity to withstand inhibition by the selection marker, which corresponds to the transformed state. The regeneration of the transgenic plant is carried out from axillary buds and newly formed leaf buds.
The term xe2x80x9cselective agentxe2x80x9d means, within the context of the invention, an agent which favours the development of the transformed cells. The selective agent used is normally kanamycin. In this case, the heterologous sequence introduced during the transformation comprises a gene encoding kanamycin resistance, for example NPT II. Kanamycin acts by blocking the chloroplast ribosomes. Consequently, a kanamycin-resistant plant is green because it is capable of making functional chloroplasts, whereas a non-resistant plant shows a yellow or white colour characteristic of the absence of functional chloroplasts. This phenomenon allows the visual selection of the transformants. The green newly formed leaf buds and the green axillary bus are selected; the principal bud, the principal plantlet and all the parts of the plant which are white or yellow are removed.
The removal of the principal bud favours the development of the axillary buds, particularly when a species exhibiting apical dominance, such as improved sunflower, is involved.
The number of selective culture cycles is preferably at least 2, including the first culture of the meristematic explant which has undergone the transformation stage. The number of cycles should be sufficient in order to allow the production of wholly transformed buds. Typically, 3 cycles should be applied, but it may be necessary, with some species or with some transformation techniques, to perform more, for example 4, 5, or 6 cycles.
In general, each stage of culture on selective medium lasts for about 15 days, but can vary between 1 and 3 weeks, depending on the species. The duration of each culture stage should be sufficient to allow the appearance of shoots having at least one leaf. If the selection cycle is repeated 3 times, the total duration of the selective culture stage will be about 6 weeks, the selective agent being present during the whole of this period. Where appropriate, a reporter gene, for example GUS, may also be incorporated during the transformation in order to allow analysis of the transformation state.
The efficiency of the method of the present invention is unexpected since the duration of the entire selective culture stages according to the invention is substantially longer than that usually applied (for example 6 weeks instead of 2 weeks). Now, it has been indicated by several authors that kanamycin has damaging effects on the regeneration of the plant (Schrammeijer et al., Plant Cell Rep. 9: 55-60, 1990). The transformation and regeneration methods developed up until now therefore tended to minimize the duration of the selection stage (Bidney et al., Proc. 13e Int. Sunflower Conf. Vol II, Pisa Sept. 1992). In fact, the present inventors have demonstrated that a prolonged period of selective culture on kanamycin exerts no inhibitory effect on the regeneration of the plant, contrary to what is indicated in the literature.
As regards the meristematic explant transformation stage, any appropriate technique can be used. This stage comprises bringing the meristematic explant into contact with Agrobacterium containing a heterologous sequence intended to be introduced into the plant cells, under conditions allowing the transfer of DNA.
There may be mentioned as example of a transformation technique the bombardment of the meristematic explant with microparticles, the bringing of the explant into contact with Agrobacterium being carried out either simultaneously, or after the bombardment. This technique makes it possible to carry out a large number of microwounds on the explant, which is necessary for the transfer of DNA by Agrobacterium. In the case of a bombardment and a simultaneous transformation, the microparticles are coated with Agrobacterium (EP-A-0,486,234). Preferably, the bringing into contact with Agrobacterium is carried out following the bombardment by applying, for example, the technique used in EP-A-0,486,233. The microparticles normally consist of gold or tungsten.
Another transformation technique consists in bringing the meristematic explant into contact with a suspension of Agrobacterium. In this case, it is preferably to make wounds on the explant, for example, by cutting it with a scalpel, in order to facilitate the transformation.
The transformation stage also comprises a period of coculturing the explant with Agrobacterium. This has a of a duration of 2 to 4 days, 3 days being preferred. The coculture medium may be any medium normally used for this purpose. A particularly advantageous medium is the M2 medium supplemented with BAP, as described in the examples below.
The Agrobacterium is normally Agrobacterium tumefaciens. Various strains may be used, for example the strain GV2260 or LBA 4404. As vector, there may be mentioned the binary plasmid pGA 492-GI, or alternatively the p35GUS intron. The xe2x80x9cstrain/vectorsxe2x80x9d pair may influence the efficiency of the transformation. It is preferable to use the strain LBA 4404 with the vector pGA 492-GI.
The nucleic acid sequence intended to be introduced into the plant cells contains any sequence capable of conferring on the plant traits of agronomic interest. Thee may be mentioned, as example, a sequence which confers resistance to insects, to herbicides or to fungal diseases (for example Sclerotinia sclerotorium or Botrytis cinerea, one or more genes for storage proteins of seeds, or which improve the quality of the storage proteins, sequences which modify the maturation of fruit, sequences which confer resistance to viruses, one or more ribozymes, one or more antisense sequences, one or more genes involved in the metabolism of fatty acids or of amino acids or one or more genes involved in male sterility.
In addition, the heterologous sequence also comprises a sequence encoding an agent which allows the selection of the transformants, for example an agent which confers resistance to an antibiotic such as kanaymcin, G418 or neomycin. The vector comprises the regulatory sequences necessary for the stable expression of the heterologous sequence. As promoter, there may be mentioned the 35S promoter, the double 35S with the translation enhancer xcexa9 or the ubiquitin promoter derived from sunflower. The NOS terminator is particularly preferred.
The transformation and selective culture stages described above form part of a set of operations which make it possible, starting with the explant, to regenerate transgenic plants. The method of the invention, in its entirety, can be summarized as follows:
2. Carrying out of the explant transformation stage as described above;
3. Carrying out of the series of selection stages as outlined above;
4. Regeneration of transgenic plants from the structures obtained at the end of the selection cycles.
According to the method of the invention, the preparation of the meristematic explant consists in a stage for preculturing an apical meristem, or an apical semimeristem, for 5 to 30 days, preferably between 5 and 8 days.
The preculture medium is a plant cell culture medium supplemented with cytokinin and more particularly with 6-benzylaminopurine (designated hereinafter as BAP). Preferably, the medium is MS (Murashige-Skoog) medium supplemented with 0.05 to 2.0 mg/l BAP, for example 0.1 mg/l of BAP, without any other hormone.
The inventors have demonstrated that the preculture stage and its duration exert a substantial influence on the development of the explant. When its duration is greater than 9 to 10 days, newly formed leaf buds appear on the leaves. The shoots regenerated on the apical meristem have, in this case, leaves which carry newly formed leaf buds on the upper epidermis.
According to this variant of the invention, it is possible, before the transformation stage, to excise the newly formed leaf buds or even simply to remove pieces of leaf, and to use them in the transformation stage. According to this embodiment of the invention, the meristematic explant subjected to the transformation may therefore consist either of part of a leaf obtained after at least 9 days of preculture capable of giving newly formed leaf buds, or of excised newly formed leaf buds.
In the case where the preculture lasts between 5 and 12 days, the apical meristem normally serves directly as meristematic explant in the transformation stage. In this case, the newly formed leaf buds are induced during the preculture stage itself, but appear only during the subsequent development, for example during the coculture or during the culture on a selective medium.
According to the invention, the same culture medium can be used for the preculture, coculture and selection stages. This medium is preferably the MS medium supplemented with BAP at a concentration of 0.05 to 2.0 mg/l, preferably 0.1 mg/l. Normally, the BAP is the only plant hormone present in the medium. In particular, it is preferable that this medium does not contain either gibberellic acid or indoleacetic acid, particularly when the preculture medium is involved. The medium may also be supplemented, during the preculture and coculture stages, with a phenolic compound, for example, acetosyringone which is capable of activating the Agrobacterium vir genes. A concentration of about 200 xcexcM is appropriate. For the selective culture stage, the medium contains at least one selective agent, advantageously kanamycin, at a concentration of between 50 and 200 mg/l, preferably 50 mg/l, and optionally, bacteriostatic agents.
The apical meristems are obtained by germination of decorticated and sterilized ripe seeds. The method of germination typically consists in culturing the seeds on a preferably solidified germination medium, for example the MS medium whose macro- and microelements are optionally reduced by half. The duration of the germination is between 2 and 4 days, preferably 4 days, at 25xc2x0 C., preferably with a photoperiod of about 16 hours.
After the preculture, transformation and selection stages, a regeneration stage is carried out starting with the wholly transformed shoots and buds. The culture media and the conditions are those usually applied in the art for the species in question. For sunflower, the regeneration conditions are indicated in the examples below.
In addition to the method of production of transgenic plants described above, the invention also relates to the newly formed leaf buds and the wholly transformed secondary meristems obtained during the method. It also relates to the transgenic plants wholly transformed in the T0 generation which are obtained from the meristematic explants.
The method of the invention exhibits numerous advantages. It is especially more efficient than the prior art techniques since the regeneration is carried out only if the explant is wholly transformed. The existing techniques involved, on the other hand, the regeneration of all the meristems which have undergone the transformation operation, followed by the selection, among the chimeric plants obtained, of those whose germinal line had been transformed. The method of the invention therefore allows a substantial economy in time and means.
Furthermore, the yield, of transgenic plants, obtained according to this method is considerably higher than that obtained according to previous techniques. For example, for sunflower, 92% of the regenerated plants give at least one transgenic plant in the progeny (Table 8 below). This figure should be compared with 0.2% to 2% which is reported by Bidney et al., Plant Molecular Biol. 18, 301-313, 1992.
The method of the invention applies to numerous plants species and more particularly to dicotyledons, especially to those which are difficult to transform and to regenerate. There may be mentioned, as example, cotton, soy bean, oleaginous plant species, for example sunflower, species belonging to the family of leguminous plants, for example pea, bean or alternatively species belonging to the Cucurbitaceae family, for example courgette. Within these species, numerous different genotypes can be used. Sunflower is particularly preferred.
Various aspects of the invention are illustrated in the figures:
FIG. 1 shows the procedure for the transformation of leaves derived from semimeristems for analyzing the expression of Glucuronidase in the axially shoots regenerated from newly formed leaf buds.
Legend:
1xe2x80x94seed
2xe2x80x94germination medium M0, 2 days
3xe2x80x94dissection of the apices: removal of the cotyledons, the radicle and the first leaf
4xe2x80x94sectioning of the apex into two halves
5xe2x80x94preculture of the semimeristems on M2 medium containing acetosyringone at 200 xcexcM for 30 days
6xe2x80x94newly formed leaf bud
7xe2x80x94axillary shoots induced from semimeristems
8xe2x80x94removal of the leaves carrying newly formed leaf buds initiated on the upper face
9xe2x80x94removal of the leaves without newly formed leaf bud
10xe2x80x94bombardment and 3 days of coculturing with Agrobacterium tumefaciens of the leaves on M2 medium containing acetosyringone at 200 xcexcM
11xe2x80x94culture on the M2 medium containing augmentin at 400 mg/l for 15 days
12xe2x80x94axillary shoots
13xe2x80x94development of newly formed leaf buds into axillary shoots
14xe2x80x94analyses of the expression of glucuronidase in the axillary shoots induced from newly formed leaf buds which appeared on the leaves
15xe2x80x94leaf explant
16xe2x80x94spots of transformed cells GUS+
17xe2x80x94transformed cell regions GUS+
18xe2x80x94transformed cell lines GUS+
19xe2x80x94transformed axillary buds GUS+
FIG. 2 shows the procedure for the transformation of semimeristems for analyzing the expression of Glucuronidase.
Legend:
1xe2x80x94seed
2xe2x80x94germination medium M0, 2 days
3xe2x80x94dissection of the apices: removal of the cotyledons, the radicle and the first leaf
4xe2x80x94sectioning of the apex into two halves
5xe2x80x94preculture of the semimeristems on M2 medium containing acetosyringone at 200 xcexcM for 5 days
6xe2x80x94bombardment and 3 days of coculturing with Agrobacterium tumefaciens of the semimeristems on M2 medium containing acetosyringone at 200 xcexcM
7xe2x80x94culture of the bombarded and cocultured semimeristems on M2 medium containing augmentin at 400 mg/l for 15days
8xe2x80x94newly formed leaf bud
9xe2x80x94axillary shoots induced from the semimeristems
10xe2x80x94axillary shoot
11xe2x80x94callus at the base
12xe2x80x94analyses of the expression of glucuronidase in the axillary shoots induced from semimeristems
13xe2x80x94spots of transformed cells GUS+
14xe2x80x94transformed cell regions GUS+
15xe2x80x94transformed newly formed leaf buds GUS+
16xe2x80x94transformed cell lines GUS+
17xe2x80x94transformed axillary buds GUS+
FIG. 3 shows the selection of axillary shoots transformed and regenerated from newly formed leaf buds on the leaves or from semimeristems.
Legend:
1xe2x80x94semimeristems bombarded and cocultured according to the procedure defined in Example 2
2xe2x80x94semimeristem-derived leaves bombarded and cocultured according to the procedure defined in Example 1
First selection culture on kanamycin:
3xe2x80x94green newly formed leaf bud resistant to kanamycin
4xe2x80x94culture of the explants on M2 medium containing augmentin at 400 mg/l and kanamycin at 50 mg/l for 15 days
5xe2x80x94non-transformed white part sensitive to kanamycin
6xe2x80x94green auxiliary bud resistant to kanamycin
7xe2x80x94transformed green part resistant to kanamycin
8xe2x80x94removal of the green axillary buds
9xe2x80x94removal of the green newly formed leaf buds
Second selection culture on kanamycin:
10xe2x80x94culture of the green axillary buds, of the green newly formed leaf buds and of the green axillary shoots on M2 medium containing augmentin at 400 mg/l and kanamycin at 50 mg/l for 15 days.
11xe2x80x94green newly formed leaf bud resistant to kanamycin
12xe2x80x94non-transformed white part sensitive to kanamycin
13xe2x80x94green axillary bud resistant to kanamycin
14xe2x80x94green part resistant to kanamycin
15xe2x80x94green axillary shoot resistant to kanamycin
16xe2x80x94removal of the green axillary buds
17xe2x80x94removal of the green newly formed leaf buds
18xe2x80x94removal of the green axillary shoots
FIG. 4 shows the regeneration of transformed plants.
Legend:
Third selection culture on kanamycin:
1xe2x80x94culture of the green axillary buds, the green newly formed leaf buds and the green axillary shoots on M2 medium containing augmentin at 400 mg/l and kanamycin at 50 mg/l for 15 days
2xe2x80x94Removal of the green axillary shoots
3xe2x80x94chimeric axillary shoot with non-transformed white parts and transformed green parts
4xe2x80x94culture in the SORBAROD system supplemented with M3 medium for 30 days in a culture chamber
5xe2x80x94taking root
6xe2x80x94washing of the M3 medium, addition of M4 medium
7xe2x80x94plant which has taken root
8xe2x80x94culture in the SORBAROD system for 10 days in the culture chamber
9xe2x80x94plant which has not taken root
10xe2x80x94development
11xe2x80x94culture in the SORBAROD system in a greenhouse for 10 days
12xe2x80x94development and acclimitization
13xe2x80x94transplanting the plants which have taken root in peat in pots, grafting the plants which have not taken root onto rootstocks, culture in a greenhouse
14xe2x80x94flowering and self-pollination
15xe2x80x94growth